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APATITE DEPOSITION DISEASE
647
Necropsy revealed massive haemorrhage into gut, myocardium, and lungs, but not brain. There was evidence of widespread monilial infection in the gut. Bone-marrow showed hypocellularity. There was nothing to suggest an alternative diagnosis to drug-induced aplastic anaemia. It seems highly probable that D-penicillamine was the drug responsible for bone-marrow failure and the
positive direct Coombs test.
We thank Dr B. E. W. Mace, Lewisham Hospital, for publish details of this patient who was under his care.
Departments of Rheumatology and Lewisham Hospital, London SE13 6LH
permission
to
ANTHONY J. RICHARDS D. S. VELVIN Hæmatology, D. N. WHITMORE E. M. WILLIAMS
APATITE DEPOSITION DISEASE
SIR,-The paper by Dr Dieppe and his colleagues (Feb. 7,
266) includes two features which can only confuse students of crystal-deposition diseases. The first is their view on the limits of size of particles of crystalline apatite which can be identified by light microscopy and hence the need for complex and costly special apparatus; the second is their use of the term "calcium hydroxyapatite". They say that "identification of apatite is difficult because the particles are usually too small to be seen by light microscopy". The diameters of the crystals identified by them, using the elegant but very expensive technique of high-resolution scanning electron-microscopy and an energy-dispersive microanalytical system, were all greater than the limit of resolution of the light microscope, being 0-35-5-0 m, except for one of 0.15 fl-m. The question of whether such crystals can be detected by light microscopy depends mainly on the amount of contrast between crystal and background, and secondarily on the limit of detection imposed by optical considerations. Contrast, as with phase-contrast microscopy, can be obtained with non-birefringent structures provided that their refractive index differs from that of the background, as would be expected with inorganic matter. With birefringent crystals, considerably greater contrast is obtained by the use of a polarising microscope. For either system of microscopy, true resolution is equal to 0.6 VN.A., so that with an objective of numerical aperture (N.A.) of 1.3and using light of 500 nm wavelength (X), the true resolution will be 0.23 p.m. On this basis only one of the nine crystals detected by Dieppe et al. would be below the limit of true resolution in the light microscope. But the ability to detect a crystal is considerably greater than the limit of resolution which, in this case, determines the smallest separation between two crystals which still allows them to be resolved as two separate crystals. Thus, provided there is sufficient contrast, it is perfectly feasible to detect a crystal of z 15 p.m diameter. Indeed in studies on xanthine crystals in muscle of xanthinurics,I and on various crystals in gout2 the birefringence characteristics of these crystals was fully measured even though they were only 0.3-0.5 p’m in diameter, and this could be done with a x 40,0-85 N.A. objective-i.e., without using the full resolution of which the polarising light microscope is capable. Moreover, Dieppe et al. showed that intradermal injection of crystalline "calcium hydroxyapatite" could produce an area of erythema around the injection site. The size and crystallinity of crystals are well known to be significant in producing such responses; the crystalline size of their hydroxyapatite was 0.5-10.0 m (diameter). Such crystals could certainly be detected by a normal polarising microscope. Although smaller aggregates of hydroxyapatite would not be detected, especially p.
if
they
were
amorphous, they
offer
no
evidence that
amor-
phous calcium phosphate hydroxide3has any irritant influence. 1 Chalmers, R. A., Watts, R. W. E., Bitensky, L., Chayen, J. J. Path. 1969, 99, 45. 2 Watts, R. W. E., Scott, J. T., Chalmers, R. A., Bitensky, L., Chayen, J. Q, Jl. Med. 1971, 40, 1. 3 Merck Index; p. 548. Rahway, New Jersey, 1968.
Dieppe et al. use the term "calcium hydroxyapatite". Rigorously "apatites" are minerals of a particular crystalline class4 (the hexagonal system) having the formula (CaF2) Ca4 (04)3’ In this sense it is self-contradictory to say that apatites are not birefringent. In studies on bone, the term is more loosely used, because the mineral component of bone is not so precisely characterised; it seems to contain many impurities in-
cluding carbonate.’ Consequently, for studying the crystal lattice of apatites, and the effect of such impurities, many workers have used the synthetic hydroxylapatite, in which OH takes the place of fluoride in the conventional apatite-i.e. CalO (OH)4 (P04)6. This may or may not be a significant component of bone. The phosphorus/calcium ratio, used by Dieppe et al. will be the same as for true apatite or for the carbonatephosphate which may also occur in bone.’ But to call this calcium hydroxyapatite is incorrect in that it implies a higher proportion of calcium in the substituted apatite than in fact occurs. It is hydroxylapatite’ or hydroxyapatite.3 Division of Cellular Biology, Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW
J. CHAYEN
*,*This letter has been shown to Dr Dieppe son, whose reply follows-ED. L.
and Dr Huskis-
SIR,-Using conventional techniques of light microscopy, of the larger particles of "apatite" are indeed visible but not identifiable. They appear as tiny specks, indistinguishable from dust and other small particles found in synovial-fluid deposits. The largest particle found in synovial fluid had a diameter of only 0.8 8 m. The greater resolution of electron microscopy was required to demonstrate the morphology of the particles, and an additional technique, such as elemental analysis, is needed for complete identification. We recognise that the term "apatite" is used with slightly different meaning by crystallographers, mineralogists and biologists. For this reason we specified the exact formula of the material used to induce experimental inflammation. The technique of X-ray energy elemental analysis which we used cannot determine with certainty the degree of substitution of fluoride or carbonate in the particle. We can, however, clearly distinguish "apatite" from the only other types of calcium and phosphate containing deposits which have been identified in biological material, including pyrophosphate. There are several analogies to be drawn from pyrophosphate-deposition disease. First the size and appearance of pyrophosphate crystals found in synovial fluids using polarised light microscopy varies considerably so that precise identification is not always possible. "Apatite" particles are 5-10 times smaller and there is again variability. Secondly pyrophosphate deposition is associated not only with acute arthritis but also with a chronic degenerative arthropathy. We suggest that "apatite" deposition may similarly be associated with a chronic degenerative arthritis. some
St Bartholomew’s Hospital, London EC1A 7BE
P. A. DIEPPE
E. C. HUSKISSON
SI UNITS AND BLOOD-PRESSURE SiR-At their recent meetings in Sydney, Australia, the Scientific Council on Hypertension of the International Society of Cardiology and the International Society of Hypertension unanimously accepted the following resolution regarding the units for the measurement of blood-pressure: "The International Society of Hypertension resolves that the millimetre of mercury (mm Hg) should be retained for blood-pressure measurement in both clinical and clinical laboratory use and Stuart, A. Crystals and the Polarising Microscope; p. 83. London, 1970. 5. Bachra, B. N. Int. Rev. connect. Tissue Res. 1970, 5, 165.
4. Hartshorne, N. H.,
Necropsy revealed massive haemorrhage into gut, myocardium, and lungs, but not brain. There was evidence of widespread monilial infection in the gut. Bone-marrow showed hypocellularity. There was nothing to suggest an alternative diagnosis to drug-induced aplastic anaemia. It seems highly probable that D-penicillamine was the drug responsible for bone-marrow failure and the
positive direct Coombs test.
We thank Dr B. E. W. Mace, Lewisham Hospital, for publish details of this patient who was under his care.
Departments of Rheumatology and Lewisham Hospital, London SE13 6LH
permission
to
ANTHONY J. RICHARDS D. S. VELVIN Hæmatology, D. N. WHITMORE E. M. WILLIAMS
APATITE DEPOSITION DISEASE
SIR,-The paper by Dr Dieppe and his colleagues (Feb. 7,
266) includes two features which can only confuse students of crystal-deposition diseases. The first is their view on the limits of size of particles of crystalline apatite which can be identified by light microscopy and hence the need for complex and costly special apparatus; the second is their use of the term "calcium hydroxyapatite". They say that "identification of apatite is difficult because the particles are usually too small to be seen by light microscopy". The diameters of the crystals identified by them, using the elegant but very expensive technique of high-resolution scanning electron-microscopy and an energy-dispersive microanalytical system, were all greater than the limit of resolution of the light microscope, being 0-35-5-0 m, except for one of 0.15 fl-m. The question of whether such crystals can be detected by light microscopy depends mainly on the amount of contrast between crystal and background, and secondarily on the limit of detection imposed by optical considerations. Contrast, as with phase-contrast microscopy, can be obtained with non-birefringent structures provided that their refractive index differs from that of the background, as would be expected with inorganic matter. With birefringent crystals, considerably greater contrast is obtained by the use of a polarising microscope. For either system of microscopy, true resolution is equal to 0.6 VN.A., so that with an objective of numerical aperture (N.A.) of 1.3and using light of 500 nm wavelength (X), the true resolution will be 0.23 p.m. On this basis only one of the nine crystals detected by Dieppe et al. would be below the limit of true resolution in the light microscope. But the ability to detect a crystal is considerably greater than the limit of resolution which, in this case, determines the smallest separation between two crystals which still allows them to be resolved as two separate crystals. Thus, provided there is sufficient contrast, it is perfectly feasible to detect a crystal of z 15 p.m diameter. Indeed in studies on xanthine crystals in muscle of xanthinurics,I and on various crystals in gout2 the birefringence characteristics of these crystals was fully measured even though they were only 0.3-0.5 p’m in diameter, and this could be done with a x 40,0-85 N.A. objective-i.e., without using the full resolution of which the polarising light microscope is capable. Moreover, Dieppe et al. showed that intradermal injection of crystalline "calcium hydroxyapatite" could produce an area of erythema around the injection site. The size and crystallinity of crystals are well known to be significant in producing such responses; the crystalline size of their hydroxyapatite was 0.5-10.0 m (diameter). Such crystals could certainly be detected by a normal polarising microscope. Although smaller aggregates of hydroxyapatite would not be detected, especially p.
if
they
were
amorphous, they
offer
no
evidence that
amor-
phous calcium phosphate hydroxide3has any irritant influence. 1 Chalmers, R. A., Watts, R. W. E., Bitensky, L., Chayen, J. J. Path. 1969, 99, 45. 2 Watts, R. W. E., Scott, J. T., Chalmers, R. A., Bitensky, L., Chayen, J. Q, Jl. Med. 1971, 40, 1. 3 Merck Index; p. 548. Rahway, New Jersey, 1968.
Dieppe et al. use the term "calcium hydroxyapatite". Rigorously "apatites" are minerals of a particular crystalline class4 (the hexagonal system) having the formula (CaF2) Ca4 (04)3’ In this sense it is self-contradictory to say that apatites are not birefringent. In studies on bone, the term is more loosely used, because the mineral component of bone is not so precisely characterised; it seems to contain many impurities in-
cluding carbonate.’ Consequently, for studying the crystal lattice of apatites, and the effect of such impurities, many workers have used the synthetic hydroxylapatite, in which OH takes the place of fluoride in the conventional apatite-i.e. CalO (OH)4 (P04)6. This may or may not be a significant component of bone. The phosphorus/calcium ratio, used by Dieppe et al. will be the same as for true apatite or for the carbonatephosphate which may also occur in bone.’ But to call this calcium hydroxyapatite is incorrect in that it implies a higher proportion of calcium in the substituted apatite than in fact occurs. It is hydroxylapatite’ or hydroxyapatite.3 Division of Cellular Biology, Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW
J. CHAYEN
*,*This letter has been shown to Dr Dieppe son, whose reply follows-ED. L.
and Dr Huskis-
SIR,-Using conventional techniques of light microscopy, of the larger particles of "apatite" are indeed visible but not identifiable. They appear as tiny specks, indistinguishable from dust and other small particles found in synovial-fluid deposits. The largest particle found in synovial fluid had a diameter of only 0.8 8 m. The greater resolution of electron microscopy was required to demonstrate the morphology of the particles, and an additional technique, such as elemental analysis, is needed for complete identification. We recognise that the term "apatite" is used with slightly different meaning by crystallographers, mineralogists and biologists. For this reason we specified the exact formula of the material used to induce experimental inflammation. The technique of X-ray energy elemental analysis which we used cannot determine with certainty the degree of substitution of fluoride or carbonate in the particle. We can, however, clearly distinguish "apatite" from the only other types of calcium and phosphate containing deposits which have been identified in biological material, including pyrophosphate. There are several analogies to be drawn from pyrophosphate-deposition disease. First the size and appearance of pyrophosphate crystals found in synovial fluids using polarised light microscopy varies considerably so that precise identification is not always possible. "Apatite" particles are 5-10 times smaller and there is again variability. Secondly pyrophosphate deposition is associated not only with acute arthritis but also with a chronic degenerative arthropathy. We suggest that "apatite" deposition may similarly be associated with a chronic degenerative arthritis. some
St Bartholomew’s Hospital, London EC1A 7BE
P. A. DIEPPE
E. C. HUSKISSON
SI UNITS AND BLOOD-PRESSURE SiR-At their recent meetings in Sydney, Australia, the Scientific Council on Hypertension of the International Society of Cardiology and the International Society of Hypertension unanimously accepted the following resolution regarding the units for the measurement of blood-pressure: "The International Society of Hypertension resolves that the millimetre of mercury (mm Hg) should be retained for blood-pressure measurement in both clinical and clinical laboratory use and Stuart, A. Crystals and the Polarising Microscope; p. 83. London, 1970. 5. Bachra, B. N. Int. Rev. connect. Tissue Res. 1970, 5, 165.
4. Hartshorne, N. H.,